1. Field of the Invention
This invention relates to a method for the preparation of antibodies which are specific for homologous hapten comprising a 3--oxybenzomorphan compound, for example, an opium alkaloid. The invention also relates to novel artificial or synthetic antigens useful for generating such specific antibodies. The antibodies produced in accordance with this invention are useful in immune assays of 3-oxybenzomorphans, in particular, morphine, heroin, codeine, and the metabolic derivatives thereof.
The immunologic terms employed herein are believed to be in accord with conventional usage and definition. Should any presently unforeseen confusion arise, unless otherwise indicated, the construction of a term shall be in accordance with its definition and usage in the well known textbook by Weiser, Myrvid and Pearsall, Fundamentals of Immunology for Students of Medicine and Related Sciences, published by Lea & Feiberger, Philadelphia, 1969.
2. Description of the Prior Art
A relatively new approach to biological assaying involves immunochemical procedures as a basis for the assay. Such procedures involve the use of antibodies which react with the compound to be assayed. A known amount of antibody and the sample, obtained from the test species, are intermixed. Theoretically, if the antibody is specific for the compound to be assayed (i.e., does not cross-react to a significant degree with biologically distinct structural homologs or analogs) one could then accurately measure the amount of antibody reacting with the test compound using conventional radioimmune or fluorescent competition assay techniques. This amount can then be translated into the amount of test compound present.
Heretofore, no immunochemical technique has been developed which produces a sufficiently reliable, reproducibly accurate opium alkaloid assay employing such immunochemical procedures. The problem, aside from the lack of sensitivity of available measurement techniques, has been that the antibodies produced in accordance with presently available methods are not sufficiently specific for the test compound. These prior art antibodies cross-react to an undersirable degree with the biologically distinct analogs or homologs of the test compound encountered in the serum sample under assay.
Antibodies to immunogenic compounds of high molecular weight, such as proteins, can be produced by administering the unaltered or natural compound to the antibody-producing host. However, small molecules which are not immunogenic by themselves, such as the opium alkaloids, must be bound to a high molecular weight immunogenic carrier. Such artificial antigens induce antibody formation. Substances which do not induce the formation of antibody, unless bound to a high molecular weight carrier, are herein termed "haptens" in conformance with conventional usage.
Conventionally, artificial antigens have been produced by conjugating immunogenic carrier molecules through the reactive functional groups on the haptenic molecule, e.g., see J. Biol. Chem., 228:713 (1957); id., 234:1090 (1958); Can. J. Biochem. Physiol., 36:1177 (1958); id., 39:941, 961 (1961); Science, 129:594 (1959); J. Immun., 92:515 (1964); Biochem., 9:331 (1970); Science, 168:1347 (1970); and J. Pharmacol., 178:253 (1971). Such attempts to produce specific antibodies by coupling carrier molecules to one of the functional groups of the hapten have successfully rendered the hapten immunogenic. However, considerable cross-reactivity is demonstrated by biologically distinct structural analogs coupled at the same or similar sites. Such non-specific binding with related compounds nugates the specificity of these conventionally produced prior art antibodies. See Steroids, 16:387 (1970); id., 18:555, 593, 605 (1971); Karolinska Symposia on Research Methods in Reproductive Endocrinology, p. 320, Ed. E. Diczfalusky Bogtrykksiut Forum, Copenhagen (1970 ); and Immunologic Methods in Steroid Determination, Eds. Peron and Caldwell, Appleton-Century-Crofts, New York (1970), 41.
Another approach to the preparation of specific antibodies to small molecules involved coupling the immunogenic carrier into the aromatic ring of the hapten. For example, see Gross, Immunochemistry, 5:55 (1968), describing the synthesis of certain immunogenic steroid-protein conjugates and the production of rabbit antiserum to beta-estradiol, coupled to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). Antisera were tested for precipitation against steroids coupled to human gamma globulin (IgG). The immunological assays employed quantitative precipitin tests, but the all-important quantitative hapten inhibition tests were not performed. This reference does report evidence of antibodies but further reports that they cross-reacted substantially. Antibody to estradiol-KLH is reported to have brought down a non-specific precipitate with testosterone-IgG. The method used for synthesizing the conjugates comprised diazotizing para-aminobenzoic acid into the steroid and then coupling the carboxy-phenylazosteroid to protein through the carboxyl group by carbodiimide condensation. The A-ring of an estrogen steroid is phenolic and, thus, azo coupling takes place adjacent the phenolic hydroxyl.
Most recently, an attempt to produce specific antibodies for 17-beta-estradiol by coupling bovine serum albumin via the alicyclic C.sub.6 position was reported in Steroids, 18:605 (1971). The reported results do not support the claimed specificity. Also, Spector et al., Science, 179:1340 (1973), have reported the preparation of antibodies to serotonin; however, the reported results indicate substantially complete cross-reactivity with the closed related analog, methoxyserotonin (i.e., 5-methoxytryptamine).
In addition to the other shortcomings of the prior art, the materials taught are not 3-oxybenzomorphans and are quite distinct from these materials. The prior art neither teaches nor suggests methods of preparing antigens useful for producing 3-oxybenzomorphan antiobdies.
To understand the importance of specificity to practical immunochemical competition assays one need only consider that a morphine assay must distinguish morphine from codeine, the commonly dispensed 3-methylmorphine, and, for that matter, from the important synthetic surrogates of morphine, such as, methadone, meperidine, and pentazocine.
Several attempts have been made to produce morphine antibodies with varying degrees of success. For example, such attempts have been reported in Hooker et al., Journal of Immunology, 38:479-490 (1940); Mingola et al., Atti. Acad. Italia, Rend. Classe Sci. Fis. Mat. Nat., (7), 2, 1103 (1941) [Chemical Abstracts, 39:1505, 1945]; Spector, U.S. Pat. No. 3,709,868, and the patent which resulted from a divisional application of that patent, U.S. Pat. No. 3,822,245; Leute et al., J.A.M.A., 221:1231 (September 1972); and Nature (New Biology) 236:93-4 (1972).
Hooker prepared, or at any rate reported, morphine and strychnine azoproteins prepared by diazotizing aminomorphine and aminostrychnine into particular, individual proteins. In each case, the amino derivatives were prepared by nitration and then reduction of the parent alkaloid. There was no evidence of morphine-specific antibody. More success was obtained with strychnine and, apparently, antibody binding specifically with strychnine was obtained. Strychnine, however, does not come within the scope of the present invention, nor is it now of pharmaceutical interest. Although strychnine is an alkaloid, and has some structural features similar to opium alkaloids, it has important differences, notably, the fact that its aromatic ring is not hydroxylated.
Hooker's failure with morphine may be explained by the fact that his starting material was not, in fact, 2-aminomorphine, as was intended. It is known to the art that nitration of morphine probably does not yield any nitromorphine.
This failure of Hooker was recognized by Mingola, who refers to the Hooker work and who, accordingly, prepared a 2-azomorphine-coupled antigen employing a different route. Mingola diazotized acetyl-p-phenylene diamine, coupled the resulting material with morphine, removed the acetyl group so as to free the second amino group, and then diazotized that compound into serum. There is no showing of the production of antibody binding with morphine, and it is doubtful that Mingola can be said to disclose such antibody. His reports regarding antibody are generally qualitative and by no means clear as to final product. The most favorable interpretation of Mingola's results would appear to be that, for both morphine and strychnine, zonal precipitation reactions were obtained between the serum recovered from rabbits after administration of the above-described antigen and the separate products of diazotizing morphine and strychnine, respectively, with other sera, presumably, sera from a different species. Apparently, several other azo proteins did not produce such precipitation reactions. Mingola provides no data to support his qualitative statements and, most importantly, there are no hapten inhibition tests, such as were done by Hooker. These tests would have provided more definitive information on the character of Mingola's products. This paper is, thus, of little value to one working in this art and would not be used by one seeking the extreme assay sensitivities required.
According to the two Spector patents, an opium alkaloid antigen is prepared by derivatizing the 3-position hydroxyl to the corresponding carboxymethyl opium alkaloid, and then coupling that derivative to a proteinic carrier by carbodiimide condensation of the carboxyl with an available amine on the protein to form an amide bond. While this material has a number of advantages, it suffers from the disabling disadvantage of having a very high cross-reactivity with codeine. Spector, in the file of his U.S. Pat. No. 3,709,868, reports experiments conducted to reproduce the work of Mingola. While Spector employed bovine serum albumin, rather than Mingola's serum, a step which should have resulted in improved results, Spector reports the antibody raised to be of poor titer and high cross-reactivity.
Leute, in his two papers, is concerned with antigens similar to those of Spector for his antibodies and assays. Again, the antigens are 3-carboxymethyl opium alkaloids coupled to a protein through the carboxyl. Again, high cross-reactivity with codeine is reported.
Thus, all of the prior art known to applicant can be summarized with the statement that the only attempts to produce an opium alkaloid antibody that have been reported employed morphine, and the quantitative data reported show that the antibody raised has an extremely high cross-reactivity with codeine.